1. Field of the Invention
The present invention concerns determination of the crystal structure of the macrophage specific receptor, CRIg (earlier referred to as STIgMA), and its complex with the C3b and C3c subunits of complement C3 (C3b:CRIg and C3c:CRIg complexes). The invention further concerns the use of the crystal structure of CRIg or the C3b:CRIg complex to screen for and identify molecules structurally and/or functionally related to CRIg, including CRIg agonists and antagonists.
2. Description of the Related Art
The complement system is a complex enzyme cascade made up of a series of serum glycoproteins, that normally exist in inactive, pro-enzyme form. C3 plays a key role in complement activation as it represents the convergence point of the three main pathways, the classical, lectin and the alternative pathways of complement. C3 interacts with more than 20 proteins (M. J. Walport, N Engl J Med 344, 1058 (2001) and (M. J. Walport, N Engl J Med 344, 1140 (2001)) via a number of distinct binding sites that are only exposed after its cleavage by the proteolytic enzyme C3 convertase (J. Janssen et al., Nature 437, 505 (2005)). The first proteolytic cleavage step generates the small (9 kDa) C3a peptide, also called anaphylatoxin, and the large (177 kDa) C3b fragment. Cleavage of C3 to C3a and C3b induces conformational changes which expose a buried thio-ester bond in C3b that can now covalently attach the molecule to particle surfaces. This process, termed “opsonization”, targets the particles for binding to macrophage complement receptors that will then clear the particle from the system.
Recently, CRIg has been identified as a receptor for C3b/iC3b opsonized particles (K. Y. Helmy et al., Cell 124, 915 (2006)). CRIg binds C3b, iC3b, as well as C3c, but is unable to form a complex with the inactive precursor C3.
In addition to its function as an opsonin, C3b is a subunit of the C3 and C5 convertases of complement. C3b first binds to the serine protease factor B (W. Vogt, G. Schmidt, B. Von Buttlar, L. Dieminger, Immunology 34, 29 (1978); Z. Fishelson, M. K. Pangburn, H. J. Muller-Eberhard, J Biol Chem 258, 7411 (1983)). This complex, after recruitment of and activation by factor D, then forms the C3bBb complex, the active C3 convertase of the alternative pathway (AP). Addition of a second C3b molecule to the C3 convertase will result in the formation of (C3b)2Bb, the C5 convertase of the alternative pathway of complement which has been shown to play a critical role in mediating inflammation in experimental animal models and human autoimmune and inflammatory diseases (M. J. Walport, K. A. Davies, B. J. Morley, M. Botto, Ann N Y Acad Sci 815, 267 (1997)). The C3b subunits of the alternative pathway convertases serve as docking sites for the respective substrate C3 or C5 and are required for catalytic activity of the convertase (N. Rawal, M. K. Pangburn, J Immunol 164, 1379 (2000). The alternative pathway of complement amplifies complement activation initiated through any of the three pathways, the alternative, classical and mannose-binding lecting pathway. As a result, deficiencies in complement factor H, a regulator of the alternative pathway, result in amplification of inflammation (N. Rougier et al., J Am Soc Nephrol 9, 2318 (1998); M. A. Abrera-Abeleda et al., J Med Genet (Nov. 18, 2005). Thus, cleavage of C3 and C5 by the alternative pathway complement convertases leads to opsonization, convertase generation and inflammation.
As a central component of the convertases, C3b serves as a target for numerous complement regulators. Among these are complement receptor 1 (CR1), membrane cofactor protein (MCP), decay accelerating factor (DAF), complement related receptor y (Crry), factor H and factor I (D. Hourcade, V. M. Holers, J. P. Atkinson, Adv Immunol 45, 381 (1989)). Binding of factor I leads to inactivation of C3b via proteolysis to form C3b and C3f (2 kDa) and finally the generation of C3dg (40 kDa), which contains the thio-ester domain (TED) and remains attached to the target, and C3c (135 kDa). A recent report on the crystal structures of C3 and C3c has significantly enhanced our understanding of the mechanism by which C3 is activated (B. J. Janssen et al., 2005, supra). C3c shows marked structural changes in comparison with C3 reflecting the consequences of multiple proteolytic cleavage steps. These structural studies suggested conformation-dependent mechanisms of C3-activation and -regulation, but do not provide an answer to the conformation of the activate species C3b.